Heparin and heparan sulfate are biologically important molecules involved in blood anticoagulation, viral and bacterial infection, angiogenesis, inflammation, cancer and development. Linhardt (2003) “Heparin: structure and activity,” J Med Chem, 46: 2551-2554. Linhardt R J, Toida T. (2004) “Role of glycosaminoglycans in cellular communication,” Acc Chem Res, 37: 431-438. Heparin finds a wide spectrum of applications including surgery, heart-lung oxygenation and kidney dialysis, treatment of deep vein thrombosis and acute coronary syndrome. Linhardt “Heparin: an important drug enters its seventh decade.” Chem. Ind. 2, 45-50 (1991); Agnelli et al. “Enoxaparin plus compression stockings compared with compression stockings alone in the prevention of venous thromboembolism after elective neurosurgery” N Engl J Med 339 (2), 80-5 (1998). Heparin is also coated on the surface of blood vessels and medical devices such as test tubes and rental dialysis machines, to form an anticoagulant surface.
Heparin is currently prepared from animal tissues in amounts of approximately 100 metric tons/year, but such heparin may be contaminated with other biological products. Linhardt Chem. Ind. 2, 45-50 (1991). Heparin contaminated with oversulfated chondroitin sulfate led to the death of nearly 100 Americans in 2008. Guerrini, et al. “Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events.” Nature Biotechnology 26, 669-675 (2008).
The natural eukaryotic heparin biosynthesis precursor, heparosan, is a polysaccharide with the repeating diassachride unit [→4) β-D-glucuronic acid (GlcA) (1→4) N-acetyl-α-D-glucosamine (GlcNAc) (1→]n, shown in FIG. 1.
Heparosan is also biosynthesized as a polysaccharide capsule in bacteria including Escherichia coli K5 and Pasteurella multicida. Lindahl U et al. (1998) “Regulated diversity of heparan sulfate” J Biol Chem 273(39):24979-24982. The initiation of K5 heparosan synthesis reportedly involves 2-keto-3-deoxyoctulosonic acid. Finke A et al. (1991) “Biosynthesis of the Escherichia coli K5 polysaccharide, a representative of group II capsular polysaccharides: polymerization in vitro and characterization of the product” J Bacteriol 173(13):4088-94. K5 heparosan is then elongated through the alternate action of the glycotransferases KfiA and KfiC that add GlcNAc and GlcA to the nonreducing end of the growing polysaccharide chain. Hodson N et al. (2000) “Identification that KfiA, a protein essential for the biosynthesis of the Escherichia coli K5 capsular polysaccharide, is an alpha-UDP-GlcNAc glycosyltransferase. The formation of a membrane-associated K5 biosynthetic complex requires KfiA, KfiB, and KfiC.” J Biol Chem 275(35):27311-5. Once synthesized, the heparosan chain is transported onto the cell surface through a pathway consisting of six proteins: KpsC, KpsD, KpsE, KpsM, KpsS and KpsT. McNulty C et al (2006) “The cell surface expression of group 2 capsular polysaccharides in Escherichia coli: the role of KpsD, RhsA and a multi-protein complex at the pole of the cell” Mol Microbiol 59(3):907-22. The K5 heparosan chain is believed to be anchored on the cell surface through lipid substitution at the reducing end of the polysaccharide to a phosphatidic acid molecule in the outer membrane of E. coli. Jann B, Jann K. (1990) “Structure and biosynthesis of the capsular antigens of Escherichia coli” Curr Top Microbiol Immunol 150:19-42. Portions of the heparosan polysaccharide can be shed from E. coli K5 through the action of K5 heparosan lyase, an enzyme originating from a bacterial phage that cleaves the heparosan chain through a β-elimination mechanism. Manzoni M et al. (1996) “Production of K5 polysaccharides of different molecular weight by Escherichia coli” Journal of Bioactive and Compatible Polymers 11(4):301-311. Manzoni M, et al. (2000) “Influence of the culture conditions on extracellular lyase activity related to K5 polysaccharide.” Biotechnology Letters 22(1):81-85. The gene encoding K5 lyase is integrated into the E. coli K5 DNA and its expression may be inducible. Manzoni M, et al. (2000). Biotechnology Letters 22(1):81-85. The activity of K5 lyase can affect the amount of heparosan released into the culture medium as well as the structure and molecular weight properties of both the cell associated and released heparosan (FIG. 1B). K5 heparosan has been estimated to have a Mw 20,000 and comprised of two major subcomponents with Mw 16,000 and 1,500. The ratio of the two subcomponents corresponds to the overall Mw and is influenced by the activity of the K5 lyase Vann W F et al (1981) “The structure of the capsular Polysaccharide (K5 Antigen) of Urinary-Tract-Infective Escherichia-Coli 010-K5-H4—a Polymer Similar to Desulfo-Heparin” European Journal of Biochemistry 116(2):359-364; Manzoni (2000) Biotechnology Letters 22(1):81-85.
Laboratory-scale studies have shown that heparosan with a weight average molecular weight (Mw)>10,000, obtained from E. coli K5 strain can be enzymatically converted to an anticoagulant polysaccharide similar to heparin. Lindahl et al. (2005) “Generation of “Neoheparin” from E coli K5 capsular polysaccharide” J Med Chem 48(2):349-352; Zhang et al. (2008) “Solution structures of chemoenzymatically synthesized heparin and its precursors” Journal of the American Chemical Society 130(39):12998-13007. Heparosan may also be used in a variety of applications (WO 2009/014559).
The present invention describes a process of E. coli K5 fermentation with a high yield of heparosan and efficient recovery of high purity heparosan suitable for industrial production of heparosan.